Monoclonal antibodies (mAbs) have become an important class of therapeutics, and there are currently approved mAbs available to treat patients suffering from various types of cancers and autoimmune disorders. The hybridoma approach (Kohler and Milstein, 1975, Nature 256:495-497) remains the most prevalent way to generate mAbs with high affinity and specificity to a target of interest. However, mAbs generated in this way are of non-human origin (usually murine) and are highly immunogenic when administered to human patients.
Several methods have been introduced in order to decrease the potential risk of immunogenicity with antibodies isolated from hybridomas, namely chimerization and humanization. Creation of chimeric antibodies, composed of murine variable regions and human constant regions (Morrison, et al., 1984, Proc Natl Acad Sci USA 81:6851-6855), was the first such method. However, since a significant portion of the antibody remains non-human, these mAbs still pose a risk of eliciting an immune response. A logical next step was the humanization, or engineering of the variable regions of these mAbs to contain more human sequence content. It was found that the murine complementarity-determining regions (CDRs), which are the principle components of the antibody that confer antigen specificity, could be “grafted” onto human frameworks (FRs) to create an antibody with higher human sequence content. This process, known as CDR-grafting (Jones, et al., 1986, Nature 321:522-525), was the first described method of antibody humanization. Since then, several methods of humanization have been described including resurfacing (Roguska, et al., 1994, Proc Natl Acad Sci USA 91:969-973), specificity-determining residue (SDR) grafting (Kashmiri, et al., 2005, Methods 36:25-34), superhumanization (Hwang, et al., 2005, Methods 36:35-42), human string content optimization (Lazar, et al., 2007, Mol Immunol 44:1986-1998), and framework shuffling (Dall'Acqua, et al., 2005, Methods 36:43-60; Damschroder, et al., 2007, Mol Immunol 44:3049-3060). The underlying assumption of all these methods is that the greater global sequence identity of the humanized sequence to a natural human sequence results in a lower risk of immunogenicity. However, due to the perceived risk of losing antigen affinity, none of these methods substantially engineer the CDRs, and as such none of these humanization methods reach the global sequence identity levels of human antibodies as they still contain mostly non-human CDRs.
More recently, “fully-human” mAbs generated from recombinant human antibody libraries (Griffiths, et al., 1994, Embo J 13:3245-3260; Knappik, et al., 2000, J Mol Biol 296:57-86) or transgenic mice comprising human germline configuration immunoglobulin gene sequences (Lonberg, 2005, Nat Biotechnol 23:1117-1125; Green, et al., 1994, Nat Genet 7:13-21; Lonberg, et al., 1994, Nature 368:856-859) have emerged as alternatives to murine generated and subsequently humanized mAbs. These mAbs have both high affinity as well as high human sequence content. Yet there remain a large number of murine antibodies with well-characterized and desirable properties. Moreover, hybridoma technology remains an accessible, efficient, and effective method for generating high quality mAbs. Thus, there is a need for efficient methods to combine the ease of creating high affinity and specificity non-human mAbs from hybridomas with the high human sequence content and expected low immunogenicity of fully-human mAbs. The current invention addresses this need.